Non-thermosensitive medium for analysing species inside a channel

ABSTRACT

The invention concerns a non-thermosensitive liquid medium for analyzing, purifying or separating species in a channel and comprising at least a polymer consisting of several polymeric segments. The invention is characterised in that the polymer is of the irregular block-copolymer or irregular comb-like polymer type and has on the average at least three junction points between polymeric segments of different chemical or topological nature. The invention also concerns methods for analyzing, purifying or separating species using a non-thermosenstive separation medium.

[0001] The present invention relates to the field of techniques for analyzing, separating and purifying species, according to which it is necessary to migrate these species in a fluid known as the “separating medium”.

[0002] The invention is directed more particularly toward proposing a separating medium that is suitable for separating species in channels or capillaries, at least one of the dimensions of which is submillimetric, and typically between 20 μm and 200 μm (referred to hereinbelow as microchannels). The invention in particular concerns methods for separating or analyzing biological macromolecules by capillary electrophoresis, by chromatography or by any method used in microchannels (capillary electrophoresis and capillary chromatography, microfluid systems, and “chip laboratories”). The invention is particularly useful in the case of electrophoresis.

[0003] In the text hereinbelow, the expression “microfluid system” will denote any system in which fluids and/or species contained in a fluid are moved inside a channel or a set of channels, at least one of the dimensions of which is submillimetric, and the term “capillary electrophoresis (CE)” will denote microfluid systems in which the transportation of species is performed by the action of an electric field.

[0004] CE and microfluid systems allow faster separations with higher resolutions than the older methods of gel electrophoresis, do not require an anticonvective medium, and their properties have been used widely to perform separations of ions in liquid medium. At the present time, the vast majority of separations of biological macromolecules performed by CE use solutions of interlocked linear water-soluble polymers that have the advantage of being able to be replaced as often as necessary.

[0005] Many noncrosslinked polymers have been proposed as media for separating species inside a channel, in particular in the context of capillary electrophoresis. The choice of the best polymer for a given application depends on several parameters. For example, for the separation of analytes as a function of their sizes, it is necessary for the medium to present the analytes with sufficiently resistant topological obstacles (Viovy et al., Electrophoresis, 1993, 14, 322). This involves the separating medium being highly interlocked, and thus relatively viscous. It is also necessary for the polymers present in the separating medium not to undergo any interactions of attraction with the analytes. The reason for this is that interactions of this type give rise to a slowing-down of certain analytes, and to additional dispersion (H. Zhou et al. HPCE 2000, Saarbrucken, 20-24/2/2000). Thus, it is well known that for DNA sequencing, or for protein separation, poorer results are obtained when the matrix has a more hydrophobic nature.

[0006] It has also been proposed in the literature to use copolymers as separating medium. In Menchen, WO 94/07133, it is proposed to use as separating medium in capillary electrophoresis, media comprising copolymers of block copolymer type which are said to be “regular” since they have hydrophilic segments of a selected and essentially uniform length and a plurality of regularly spaced hydrophobic segments, at a concentration higher than the overlap concentration between polymers. These media have the advantage of being shear-thinning, i.e. they can be introduced into a capillary under high pressure, while at the same time presenting solid topological obstacles in the absence of external pressure. Unfortunately, the media that may be used according to this principle are difficult to synthesize, which makes them expensive and limits the type of structures that may be envisaged. Also, these polymers are relatively hydrophobic, and their performance qualities for DNA sequencing, for example, are mediocre.

[0007] It has also been proposed to use as separating media thermosensitive media, the viscosity of which varies greatly during an increase in temperature. This type of medium has the advantage of allowing the injection of said medium into the capillary at a first temperature in a state of low viscosity, and the separation at a second temperature in a state of higher viscosity that displays good separation efficiency, as is commonly performed in gel electrophoresis, in particular with agarose. Patent applications WO 94/10561 and WO 95/30782 especially propose media that allow an easier injection by raising the temperature. In point of fact, said patent applications essentially describe microgels capable of decreasing in volume at high temperature (thus leading to a dilute solution of discontinuous particles of low viscosity) and of swelling at low temperature until they entirely fill the separating channel (thus giving the medium a gelled nature and good separating properties). Patent application WO 98/10274 itself proposes a molecular separating medium comprising at least one type of block copolymers that is in solution at a first temperature and in a gel-type state at a second temperature. The media described comprise triblock polymers of low molecular masses (typically less than 20 000), of the polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE) family and more specifically (POE₉₉-POP₆₉-POE₉₉ in which the indices represent the number of monomers of each block) (trade name “Pluronic F127”). At low temperature, the two POE segments at the ends of the triblock systems are water-soluble and, given the low molecular mass of the copolymer, the solutions are relatively nonviscous up to a high concentration. By raising the temperature by about 15-25° C., the central POP segment becomes more hydrophobic, and these polymers become associated to form a gel-type state. However, this mechanism presents several drawbacks in electrophoresis. Firstly, it gives rise to a gel state that has good electrophoretic separating properties only at high polymer concentrations, of greater than 15 g/100 ml or even 20 g/100 ml, which leads to high friction and long migration times. Moreover, the dependence of the properties as a function of the rate of change of temperature makes the reproducibility of the results random. Finally, for many applications and in many devices, it is inconvenient, or even impossible, to change the temperature between the stage of filling of the channel and the separating stage.

[0008] In Madabhushi, U.S. Pat. No. 5,552,028, WO 95/16910 and WO 95/16911, it is also proposed to use separating media comprising a screening medium and a surface-interaction component consisting of a polymer with wall-adsorption properties, with a molecular mass of between 5 000 and 1 000 000, of the disubstituted acrylamide polymer type. These matrices, and more particularly polydimethylacrylamide (PDMA), make it possible to reduce the electroosmosis and in certain applications, for instance sequencing, lead to good separating properties. However, they are relatively hydrophobic, which limits their performance qualities for certain applications, for instance DNA sequencing, and is even more harmful for other applications, for instance protein separation. Moreover, they lead to slow separations.

[0009] Consequently, despite the large number of studies and systems proposed, a medium that is optimum for all the various aspects of cost, of separation efficiency, of reduction of interactions with the walls and of convenience of use is not available at the present time for all the applications mentioned above.

[0010] One object of the present invention is, precisely, to propose the use of a family of polymers that is particularly advantageous as non-thermosensitive liquid separating medium for the separation, analysis or purification of species in channels.

[0011] More particularly, a subject of the present invention is a non-thermosensitive liquid medium for analyzing, purifying or separating species inside a channel, and comprising at least one polymer composed of several polymer segments, characterized in that said polymer is of the irregular block copolymer type or irregular comb polymer type and has on average at least three junction points established between polymer segments of different chemical or topological nature.

[0012] For the purposes of the invention, the term “polymer” denotes a product consisting of a set of macromolecules and characterized by certain properties such as molecular mass, polydispersity, chemical composition and microstructure. The polydispersity characterizes the molecular mass distribution of the macromolecules, in the meaning of the mass-average familiar to those skilled in the art. The term “microstructure” means the way in which the monomers forming part of the chemical composition of the macromolecules are arranged within the latter.

[0013] According to the invention, the term “liquid” means, as opposed to a “gel”, any condensed medium capable of flowing, whether it is newtonian or viscoelastic.

[0014] In the present case, gels derived from the copolymerization of monomers in the presence of difunctional or multifunctional crosslinking agent(s) are excluded from the field of the invention. The reason for this is that, given their crosslinked state, these gels are solid or elastic and are therefore not liquid. In particular, they are unsuitable for introduction into a capillary.

[0015] The liquid medium according to the invention is non-thermosensitive, i.e. it does not display, between its solidification point plus 10° C., and its boiling point minus 10° C., a sudden change in its viscosity. The term “sudden change” means a variation by a factor of 2 or more, over a temperature range of 20° C. or less.

[0016] For the purposes of the invention, the expression “separation method” is intended to cover any method directed toward separating, purifying, identifying or analyzing all or some of the species contained in a sample. In this case, the liquid is referred to as the “separating medium” and through it pass the species to be separated or at least some of them in the course of the separation process.

[0017] The term “species” is generally intended to denote particles, organelles or cells, molecules or macromolecules, and in particular biological macromolecules, for instance nucleic acids (DNA, RNA or oligonucleotides), nucleic acid analogs obtained by synthesis or chemical modification, proteins, polypeptides, glycopeptides and polysaccharides. In analytical methods, said species are commonly referred to as “analytes”.

[0018] The invention is particularly advantageous in the case of electrokinetic separation methods.

[0019] The term “electrokinetic separation” is intended to cover any method directed toward separating all or some of the species contained in a mixture by making them migrate in a medium by the action of an electric field, whether the field exerts its motor action on the analytes directly or indirectly, for example by means of a displacement of the medium itself, for instance in electrochromatography, or by means of a displacement of associated species such as micells, in the case of micellar electrochromatography, or by any combination of direct and indirect actions. Any separation method in which said action of the electric field is combined with another motor action of nonelectric origin are also considered as an electrokinetic separation method according to the invention. Accordingly, methods of capillary electrophoresis or of electrophoresis on “chips” are referred to as “electrokinetic”.

[0020] Advantageously, in particular in the case of electrokinetic separations, the liquid will consist of an electrolyte.

[0021] For the purposes of the invention, the term “electrolyte” denotes a liquid capable of conducting ions. In the most common case, this medium is a buffered aqueous medium, for instance buffers based on phosphate, tris(hydroxymethyl)aminomethane (TRIS), borate, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), histidine, lysine, etc. Numerous examples of buffers that may be used in electrophoresis are known to those skilled in the art, and a certain number of them are described, for example, in Sambrook et al., “Molecular Cloning: a laboratory manual”, Cold Spring Harbor Lab, New York, 1989. However, any type of electrolyte may be used in the context of the invention, especially aqueous-organic solvents such as, for example, water-acetonitrile, water-formamide or water-urea mixtures, or polar organic solvents such as, also by way of example, N-methylformamide. The “sequencing buffer” electrolytes consisting of an aqueous buffer at alkaline pH containing an appreciable proportion of urea and/or of formamide are found to be particularly useful in the context of the invention.

[0022] The term “channel” denotes any volume delimited by one or more solid walls, having at least two orifices and intended to contain a fluid or to have a fluid pass through it.

[0023] The invention is particularly advantageous in systems comprising at least one channel, at least one dimension of which is submillimetric, such as capillary electrokinetic separation systems, microfluid systems and, more generally, systems for separating species using microchannels.

[0024] According to one preferred variant, the invention is directed toward the use, as a liquid separating medium, of a solution containing polymers having on average at least four junction points, preferably a number of junction points of between 4 and 100 and more preferably a number of junction points of between 4 and 40.

[0025] The term “junction point” means a point connecting either two polymer segments of significantly different chemical nature, for instance in the case of a block copolymer, or a point of crosslinking between more than two polymer segments of identical or different chemical nature, for instance in comb polymers. By way of example, a comb polymer bearing three side branches comprises three junction points and seven separate polymer segments. Similarly, a sequential block copolymer of A-B-A-B type comprises three junction points and four separate polymer segments.

[0026] For the purposes of the invention, the terms “polymer segment” and “segment” denote a set of monomers linked together in a linear and covalent manner, and belonging to a given type of chemical composition, i.e. having specific overall physicochemical properties, in particular as regards the salvation, the interaction with a solid wall, a specific affinity toward certain molecules, or a combination of these properties.

[0027] An example of a polymer segment for the purposes of the invention is given by the sequence, within a copolymer, of monomers that are all identical (homopolymer segment), or a copolymer that has no significant composition correlation over distances of more than a few monomers (segment of random copolymer type). The polymer according to the invention is composed of several “different” polymer segments. Two polymer segments that differ in their chemical nature and/or their topology, i.e. the spatial distribution of the segments relative to each other, for example skeleton as opposed to side branch, are different for the purposes of the invention.

[0028] According to a first preferred variant, the polymers according to the invention are of the irregular block copolymer type.

[0029] For the purposes of the invention, the term “block copolymer” denotes a copolymer consisting of several polymer segments linked together covalently, and belonging to at least two different types of chemical composition. Thus, two adjacent polymer segments within a linear block copolymer are necessarily of significantly different chemical nature. The block copolymer is defined by the fact that each of the segments comprises a sufficient number of monomers to have within the separating medium physicochemical properties, and in particular in terms of salvation, that are comparable to those of a homopolymer of the same composition and of the same size. This is in contrast with a random copolymer, in which the various types of monomer follow each other in an essentially random order, and give the chain locally overall properties that are different from those of homopolymers of each of the species under consideration. The size of the homopolymer segments required to obtain this block nature may vary as a function of the types of monomers and of the electrolyte, but it is typically a few tens of atoms along the skeleton of said segment. It should be noted that it is possible to make a block copolymer within the meaning of the invention, in which all or some of the segments themselves consist of a copolymer of random type, insofar as it is possible to distinguish within said block copolymer polymer segments of size and of difference in chemical composition that are sufficient to give rise from one segment to another to a significant variation in the physicochemical properties, and in particular in the salvation. In particular, in order to be considered as a “polymer segment” within the meaning of the invention, a portion of polymer must comprise along its skeleton at least 10 atoms.

[0030] According to one preferred mode of this variant, the polymer according to the invention is of the irregular sequential block copolymer type.

[0031] For the purposes of the invention, the term “sequential block copolymer” means a block copolymer composed of polymer segments belonging to at lest two different chemical types, linked together in a linear manner.

[0032] According to a second preferred variant, the polymer according to the invention is of the irregular comb polymer type.

[0033] For the purposes of the invention, the term “comb polymer” denotes a polymer having a linear skeleton of a certain chemical nature, and polymer segments known as “side branches”, of identical or different chemical nature, which are also linear but significantly shorter than the skeleton, and are covalently attached to said skeleton via one of their ends. In a comb polymer, the polymer segments constituting the skeleton and those constituting the side branches differ in their topological nature. If the polymer segments constituting the side branches of the comb polymer and those constituting its skeleton also differ in their chemical nature, the polymer simultaneously has the characteristic of a “comb polymer” and that of a “block copolymer”. Such polymers, which are known as “comb copolymers”, constitute a subset of comb polymers and can, of course, be used in the context of the invention.

[0034] Needless to say, the combined use of block copolymer(s) and comb polymer(s) in a medium in accordance with the invention may be envisaged.

[0035] The number of polymer segments of a given chemical or topological type present in the polymers according to the invention is understood as being an average value, it being understood that it is always a matter of a population of a large number of molecules having in said numbers a certain polydispersity.

[0036] In the present description and unless otherwise mentioned, all the molecular masses and also all the averages for all the chains or all the polymer segments, for instance the average molecular mass, or the average number of atoms along the skeleton, the number of junction points, or the average number of grafts in the case of a comb polymer, are understood as being mass averages within the usual meaning of polymer physics.

[0037] All the polymers under consideration according to the invention, namely block copolymers or comb polymers, also have the advantageous characteristic of being of irregular type, i.e. all the segments of at least one type of chemical or topological nature forming part of their composition have a polydispersity of at least 1.5 and preferably greater than 1.8.

[0038] The polydispersity of a type of polymer segment forming part of the composition of a polymer according to the invention is understood as being the average value of the molecular mass of said segments, taken over all the segments of this type forming part of the composition of said polymer (mass average within the usual meaning of polymer physicochemistry).

[0039] One preferred variant of an irregular comb polymer consists in displaying a polydispersity of the side branches of at least 1.5 and preferably greater than 1.8.

[0040] Another preferred variant of an irregular comb polymer consists in displaying a polydispersity of the segments of the skeleton included between two side branches of at least 1.5 and preferably greater than 1.8.

[0041] In another preferred embodiment, the segments of each of the types of chemical or topological nature forming part of the composition of the polymers according to the invention have a polydispersity of at least 1.5 and preferably greater than 1.8.

[0042] According to one preferred embodiment, the polydispersity of the polymers according to the invention is greater than 1.5 and preferably greater than 1.8.

[0043] The length and number of the different polymer segments present in the comb polymers or the copolymers used in the media according to the invention, and also the chemical nature thereof, may vary significantly in the context of the invention, and the properties of said media may thus be varied widely depending on the desired application, as will be shown more specifically in the description of the implementation examples.

[0044] According to one preferred embodiment, the polymers according to the invention have a molecular mass (mass-average) of greater than 50 000, preferably greater than 300 000, more preferably greater than 1 000 000 and better still greater than 3 000 000.

[0045] According to one preferred embodiment, said polymers according to the invention show within the separating medium significant affinity for the walls of said channel.

[0046] One particularly preferred mode consists in presenting within the polymer according to the invention at least one type of polymer segment showing, within the separating medium, specific affinity for the wall, and at least one type of polymer segment showing in said medium less or no affinity for the wall.

[0047] The presence of polymer segments of this type allows the medium according to the invention to reduce the adsorption of species onto the walls of the channel and/or the electroosmosis.

[0048] Specifically, one problem for all the methods involving species within channels is the adsorption of said species onto the walls of said channels. This problem is particularly exacerbated in the case of small channels and biological macromolecules, the latter often being amphiphilic. This phenomenon of adsorption onto walls of species contained in the sample or the fluid has the consequence of retarding certain analytes and of creating an additional dispersion, and thus a loss of resolution, in the case of analytical methods. This adsorption may also give rise to a certain amount of contamination of the walls of the channel, which may affect the fluids that it is desired to introduce thereafter into said channel.

[0049] Another limitation, which more particularly relates to electrokinetic separation methods, is electroosmosis, a movement as a whole of the separating medium due to the presence of charges on the walls of the capillary or the channel. Since this movement is often variable over time and nonuniform, it is detrimental to the reproducibility of the measurements and to the resolution. It is caused by the charges that may be present on the surface of the capillary due to its chemical structure, but may also be created or increased by the adsorption, onto the wall, of charged species initially contained in the samples to be separated, and in particular proteins.

[0050] The polymers according to the invention of the type containing polymer segments of at least one type showing within the separating medium specific affinity for the wall, have, on account of the presence of a plurality of segments of this type, and on account of the relatively high average molecular mass of said segments, a high adsorption energy, and thus reduce the electroosmosis in a long-lasting manner. Moreover, since the polymers according to the invention also comprise in their structure polymer segments that show in said medium less or no affinity for the wall, they avoid an excessively hydrophobic nature that is harmful for resolution, and can more efficiently repel the analytes from the walls.

[0051] Typically, types of polymer segments that show no affinity for the wall consist of polymers that show good solubility in the separating medium. However, there may be polymers which are soluble in said medium, but which nevertheless show therein particular affinity for a wall. When the separating medium is an aqueous solution, segments with no affinity for the wall are typically highly hydrophilic segments. On the other hand, segments with affinity are relatively nonhydrophilic, or even hydrophobic. Needless to say, other more specific types of affinity may be used, depending on the nature of the wall and that of the separating medium.

[0052] Copolymers that are optimized for performing the invention are especially those in which all the segments that have specific affinity for the wall represent between 2% and 80% by mass and preferably between 5% and 30% of the total average molar mass of said copolymers, or between 3% and 85% and preferably between 5% and 50% of the total composition of the copolymers in terms of number of moles of monomers.

[0053] Another preferred embodiment, which is particularly advantageous when the analytes are biological macromolecules, consists in using polymers according to the invention that also show specific affinity for one or more analytes.

[0054] This affinity may be obtained by incorporating into the structure of said polymers polymer segments capable of showing specific affinity for certain analytes. Such polymer segments may consist, for example, and in a nonexhaustive manner, of a predetermined sequence of different monomers, for instance a polynucleotide or a polypeptide. This affinity may also be obtained by combining with the polymer according to the invention a native or denatured protein, a protein fraction or a protein complex, or alternatively an acidic or basic function, and/or a function of Lewis acid or Lewis base type.

[0055] As illustrations of the various structures that may be adopted by the copolymer according to the invention, mention may be made most particularly of those in which all or some of said copolymer is:

[0056] in the form of irregular sequential block copolymers. In this case, one preferred variant consists in alternating, along the polymer, segments with specific affinity for the wall, and segments with reduced or no affinity for the wall. It may also be envisaged to alternate, along the polymer, segments showing specific affinity for certain analytes, and segments showing reduced or no affinity for said analytes;

[0057] in the form of irregular comb copolymers. In this case, one preferred variant is characterized in that said polymers are in the form of comb polymers whose skeleton consists of several polymer segments that show specific affinity with the wall, and the side branches of which consist of polymer segments showing reduced or no affinity for the wall, or comb polymers whose side branches consist of polymer segments showing specific affinity for the wall, and whose skeleton consists of polymer segments showing reduced or no affinity for the wall. These polymers may also be in the form of comb polymers, certain side branches of which consist of polymer segments showing specific affinity for certain analytes, and the skeleton of which consists of polymer segments showing reduced or no affinity for these analytes.

[0058] Needless to say, systems in which several types of preferred variants above are combined together, either by combining polymer segments of more than two different types, or in the form of a mixture of different copolymers, also fall within the scope of the invention. It is thus possible, for example, to combine within a copolymer according to the invention polymer segments showing affinity for the wall, polymer segments or groups showing specific affinity for certain analytes, and polymer segments showing no specific affinity either for the walls or for the analytes. It is also possible, again by way of example, to combine in a medium according to the invention block copolymers comprising polymer segments showing affinity for the wall and polymer segments showing no specific affinity either for the walls or for the analytes, and polymers comprising polymer segments or groups showing specific affinity for certain analytes, and polymer segments showing no specific affinity either for the walls or for the analytes.

[0059] In one preferred mode of the invention, all of the polymer segments of a given type of chemical or topological nature have on average along their skeleton a number of atoms of greater than 75, and more preferably greater than 210, or have a molecular mass of greater than 1 500 and preferably greater than 4 500.

[0060] According to an even more preferred embodiment, the various types of segments have along their skeleton an average number of atoms of greater than 75, and more preferably greater than 210, or have a molecular mass of greater than 1 500 and preferably greater than 4 500.

[0061] According to one preferred embodiment, the separating medium consists of a liquid in which at least one polymer in accordance with the invention is dissolved to a proportion of from 0.1% to 20% and preferably from 1% to 6% by weight.

[0062] It is particularly advantageous for implementing the invention to use block copolymers or comb homopolymers in which at least one of the types of segments consists of a polymer chosen from polyethers, polyesters, for instance polyglycolic acid, soluble random homopolymers and copolymers of the polyoxyalkylene type, for instance polyoxypropylene, polyoxybutylene or polyoxyethylene, polysaccharides, polyvinyl alcohol, polyvinylpyrrolidone, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyoxazoline, polystyrene sulfonate, and substituted or unsubstituted acrylamide, methacrylamide and allyl polymers and copolymers.

[0063] As representatives of the types of polymer segments showing, in an aqueous separating medium, little or no affinity with the walls, mention may be made most particularly of polyacrylamide and polyacrylic acid, polyacryloylaminopropanol, water-soluble acrylic and allylic polymers and copolymers, dextran, polyethylene glycol, polysaccharides and various cellulose derivatives such as hydroxyethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or methylcellulose, polyvinyl alcohol, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyoxazoline, polystyrene sulfonate, and also polymers bearing hydroxyl groups, and all the random copolymers of the derivatives mentioned above.

[0064] Needless to say, other polymer segments that are soluble in the separating medium may be used according to the invention, as a function of the nature of said fluid and of that of the walls of the channel, the particular application and the ease of introducing them into a block polymer of the desired structure.

[0065] As representatives of the polymer segments, which may or may not be soluble in aqueous solvents, and which may show therein particular affinity for the walls, mention may be made of dimethylacrylamide, acrylamides N-substituted with alkyl functions, acrylamides N,N-disubstituted with alkyl functions, allyl glycidyl ether, copolymers of the above acrylic derivatives with each other or with other acrylic derivatives, alkanes, fluoro derivatives, silanes, fluorosilanes, polyvinyl alcohol, polymers and copolymers involving oxazoline derivatives, and also in general polymers that have a combination of carbon-carbon bonds, ether-oxide functions and epoxide functions, and also all the random copolymers of these compounds.

[0066] Many types of polymer segments may be chosen to make up the polymer segments constituting a polymer according to the invention, as a function of the envisaged electrolyte, from all the types of polymers known to those skilled in the art, in particular from those soluble in aqueous medium. Reference may thus be made to the book “Polymer Handbook” Brandrupt & Immergut, John Wiley, New York.

[0067] The polymers according to the invention may be natural or synthetic. According to one preferred variant for the variety and control that it allows with regard to the microstructure, the polymers according to the invention are synthetic polymers.

[0068] The following are most particularly suitable for the invention:

[0069] copolymers of the comb copolymer type, the skeleton of which is of dextran, acrylamide, acrylic acid, acryloylaminoethanol or (N,N)-dimethylacrylamide type and onto which are grafted side segments of acrylamide, substituted acrylamide or (N,N)-dimethylacrylamide (DMA) type, or of the DMA/allyl glycidyl ether (AGE) copolymer type, or alternatively of homopolymer or copolymer of oxazoline or of oxazoline derivatives;

[0070] non-thermosensitive copolymers of the irregular sequential block copolymer type having along their skeleton an alternation of segments of polyoxyethylene type and of segments of polyoxypropylene type, or an alternation of segments of polyoxyethylene type and of segments of polyoxybutylene type, or more generally an alternation of segments of polyethylene and of segments of polyether type that are appreciably more hydrophobic than polyoxyethylene;

[0071] copolymers of the irregular sequential block copolymer type having along their skeleton an alternation of segments of acrylamide, acrylic acid, acryloylaminoethanol or dimethylacrylamide type, on the one hand, and segments of (N,N)-dimethylacrylamide (DMA) type, or of DMA/allyl glycidyl ether (AGE) copolymer type, or alternatively of homopolymer or copolymer of oxazoline or of oxazoline derivatives;

[0072] polymers of the irregular comb polymer type, the skeleton of which is of agarose, acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type, and onto which are grafted side segments of agarose, acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type;

[0073] copolymers of the irregular comb copolymer type, the skeleton of which is of the acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of agarose, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type, and bears short-chain hydrophobic side segments such as alkyl chains, aromatic derivatives, fluoroalkyls, silanes or fluorosilanes.

[0074] It should also be noted that, in most applications, it is preferable to use a polymer according to the invention that is essentially neutral. However, it may be useful for certain applications, and in particular to avoid the adsorption of species containing both charges and hydrophobic portions, to select a polymer according to the invention that is deliberately charged, preferably opposite in charge to that of said species.

[0075] As regards the preparation of the copolymers used according to the invention, it may be carried out by any conventional polymerization or copolymerization technique. The choice of preparation method is generally made by taking into account the structure desired for the copolymer, i.e. comb or linear structure, and the chemical nature of the various blocks of which it is made.

[0076] As representatives of these preparation variants, mention may be made most particularly of processes according to which said copolymers are obtained by:

[0077] polycondensation, ionic or free-radical polymerization or copolymerization of identical or different monomers, of identical or different macromonomers, or of a mixture of identical or different monomers and macromonomers, or

[0078] by grafting several polymer segments onto a linear or branched polymer skeleton of identical or different chemical nature.

[0079] Preferably, all or some of the copolymers used according to the invention are obtained by

[0080] a: copolymerization of monomers and macromonomers comprising a reactive function at at least one of their ends, or

[0081] b: copolymerization of macromonomers comprising at least one reactive function in their structure.

[0082] For the purposes of the invention, the term “reactive function” means a group that allows the molecule bearing this group to be incorporated into the macromolecule during the copolymerization reaction without interrupting said copolymerization.

[0083] With the aid of the rules and preferred modes listed above, a person skilled in the art is capable of preparing the copolymers in accordance with the invention, by adapting the structure, the nature and the mode of preparation of said polymers as a function of the desired separation properties for one application or another.

[0084] A subject of the present invention is also a process for separating, analyzing and/or identifying species contained in a sample, characterized by performing the following steps:

[0085] a/ filling the channel of a separating device with separating medium according to the invention,

[0086] b/ introducing said sample containing said species into one end of said channel,

[0087] c/ applying an external field intended to move certain species contained in the sample, especially an electric field, and

[0088] d/ recovering or detecting the passage of said species at a point along the channel that is different from the point of introduction of the sample.

[0089] In one preferred variant, it is not necessary to change the temperature between the capillary filling stage and the analysis stage.

[0090] Depending on the particular applications, the separating medium may contain, besides the polymers according to the invention, other elements, and in particular components that interact with the species or the walls. Many elements of this type are known to those skilled in the art.

[0091] In the present case, it is possible to combine in the separating medium polymers of the irregular block copolymer type, and other polymers capable of interacting with analytes either by steric interaction or by affinity, in order to improve the performance qualities compared with those obtained with the polymer according to the invention used alone. In this case, polymers that are more particularly preferred according to the invention are those having a mass fraction of polymer segments showing specific affinity for the wall that is greater than when these polymers are used alone. This fraction may be between 20% and 80%.

[0092] A subject of the present invention is also the use of a separating medium according to the invention for separating, purifying, filtering or analyzing species chosen from molecular or macromolecular species, and in particular biological macromolecules, for instance nucleic acids (DNA, RNA or oligonucleotides), nucleic acid analogs obtained by synthesis or chemical modification, proteins, polypeptides, glycopeptides and polysaccharides, organic molecules, synthetic macromolecules or particles such as mineral particles, latices, cells or organelles.

[0093] In the case of electrophoresis analysis methods, the invention is particularly useful for DNA sequencing, for which it allows minimum bandwidths to be obtained. It is also particularly favorable for separating proteins, proteoglycans, or cells, for which the problems of adsorption onto the wall are a particular handicap and particularly difficult to solve.

[0094] Advantageously, the claimed medium may be used in a channel of which at least one dimension is of submillimetric size.

[0095] As regards the apparatus, the claimed medium is particularly advantageous for microfluidic systems, since it makes it possible, by means of an optimum choice of the various types of blocks within the polymers, to combine blocks that show good affinity for the surface of the channel in order to obtain a long-lasting treatment, and blocks that show good repulsion for the species to be separated, irrespective of said species and of the chemical nature of said channel.

[0096] The media according to the invention and the separation methods using these media are particularly advantageous for electrophoretic separation and diagnostic applications, gene typing, and large-throughput screening, quality control, or for detecting the presence of genetically modified organisms in a product.

[0097] In point of fact, the polymers of which the separating medium under consideration in the context of the present invention is composed are found to be advantageous in several respects.

[0098] Firstly, their capacity to display “block polymer” nature allows them to combine properties belonging to polymers of different chemical nature, and that cannot always be united in a homopolymer or a random copolymer. They thus make it possible to more flexibly adapt the chemical nature of the separating medium, firstly as a function of the species to be separated, and secondly as a function of the chemical nature of the channels in which the separation is performed. They are thus particularly advantageous both in applications using channels consisting of polymers or elastomers such as PDMS (polydimethylsiloxane), PMMA (polymethyl methyacrylate), polycarbonate, polyethylene, polypropylene, polyethylene terephthalate or polyimide, or of mineral materials such as glass, ceramics, silicon, stainless steel or titanium, and in more traditional applications using channels whose walls are made of fused silica.

[0099] Compared with the block copolymers of the prior art, the polymers according to the invention also show superior performance qualities in terms of resolution, which is most probably associated with their irregular nature, i.e. the polydispersity of the polymer segments forming part of the polymers according to the invention. This characteristic is particularly surprising, since the set of block copolymers used in the prior art deliberately involves copolymers containing regularly spaced segments and/or having a selected and essentially uniform length (i.e. low polydispersity). This polydispersity of the segments, in the polymers according to the invention, also shows advantages in terms of cost and flexibility in formulating, since polymers comprising such polymolecular segments are not only more efficient, but also easier to prepare. In particular, they may be prepared with high molecular masses.

[0100] In the applications for which a reduction of electroosmosis or interaction of species with the wall is desired, the polymers according to the invention have, on account of the presence in their structure of a large number of polymer segments that show significant affinity for the wall, high adsorption energy and can thus reduce the electroosmosis and the adsorption of species in a long-lasting manner.

[0101] Finally, it is also very likely that the combination of a linear skeleton and of a plurality of junction points gives the separating media according to the invention some of the properties of gels at the local scale, which is beneficial in terms of separation efficiency, while at the same time conserving them at the large scale, and in particular as regards the flow properties, properties that are comparable with those of linear polymers.

[0102] The figures and examples given below are presented as nonlimiting illustrations of the present invention.

FIGURES

[0103]FIG. 1: Example of diagrammatic configuration 1 a: of an irregular sequential block copolymer; 1 b: of an irregular comb polymer; 1 c: of an irregular comb copolymer. The bold lines correspond to one type of chemical nature, and the fine lines to another type of chemical nature.

[0104]FIG. 2: Control electrophoregram representing the separation of the Pharmacia Biotech 50-500 bp sizer, obtained at 50° C. in an ABI 310 machine (Perkin Elmer), using an untreated capillary and, as separating medium, a 100 mM Na TAPS, 2 mM EDTA, 7M urea buffer, in which is dissolved 5% of a commercial homopolymer of the polyacrylamide type (molecular mass 700 000-1 000 000); the DNA sizes corresponding to the various peaks are indicated on the diagram, as number of bases.

[0105]FIG. 3: Control electrophoregram representing a separation under conditions identical to those of FIG. 2, with a “POP6” commercial separating medium from PE Biosystems. The DNA sizes corresponding to the various peaks are indicated on the diagram, as number of bases.

[0106]FIG. 4: Electrophoregram representing a separation under conditions identical to those of FIG. 2, with a 100 mM Na TAPS, 2 mM EDTA, 7M urea separating medium, in which is dissolved 5% of P(AM-PDMA)-2 comb copolymer described in Example 2. The DNA sizes corresponding to the various peaks are indicated on the diagram, as number of bases.

[0107]FIG. 5: Comparison of the resolution calculated between peaks differing from one base to 500 bases, obtained at 50° C. in an ABI 310 machine (Perkin-Elmer), using as separating medium:

[0108] a: a “Pop6” commercial separating medium from PE Biosystems,

[0109] b: a 50 mM Na TAPS, 2 mM EDTA, 7M urea buffer, in which is dissolved 5% of linear acrylamide (molecular mass 700 000˜1 000 000),

[0110] c: the same buffer, in which is dissolved 5% of irregular block copolymer according to the invention P(AM-PDMA)-2 described in Example 2.

[0111]FIG. 6: Viscosity of solutions at 3% of linear acrylamide (LPA) and of the copolymers according to the invention poly(AM-PDMA)-1, prepared according to Example 2, poly(AM-PDMA)-2, prepared according to Example 4, and poly(AM-PDMA)-3, prepared according to Example 5.

[0112]FIG. 7: Resolutions obtained during the electrophoretic separation of DNA, in solutions of linear acrylamide (LPA), of commercial polymer (POP5), and of the copolymers according to the invention poly(AM-PDMA)-1, prepared according to Example 2, poly(AM-PDMA)-2, prepared according to Example 4, and poly(AM-PDMA)-3, prepared according to Example 5, at 3% and 5%.

EXAMPLE 1

[0113] Preparation of a functionalized PDMA macromonomer with a molecular mass in the region of 10 000, for the preparation of copolymers in accordance with the invention.

[0114] 1) Polymerization of PDMA

[0115] The free-radical polymerization of N,N-dimethylacrylamide (DMA) is performed in pure water. The initiator is a redox couple for which the oxidizing agent is potassium persulfate K₂S₂O₈ (KPS) and the reducing agent is aminoethanethiol AET.HCl. The initiation reaction is:

[0116] K₂S₂O₈+2Cl⁻, NH₃ ⁺—CH₂CH₂—SH→2KHSO₄+2Cl⁻, HN₃ ⁺—CH₂—CH₂—S⁺

[0117] AET.HCl also acts as transfer agent, which allows the chain length to be controlled.

[0118] Procedure

[0119] 0.18 mol of DMA and 200 ml of water are placed in a 500 ml three-necked flask on which is mounted a condenser, and equipped with a nitrogen inlet device. The mixture is then stirred and heated to 29° C. with a water bath. Sparging with nitrogen is commenced. After 45 minutes, 0.61 g of AET.HCl (0.0054 mol) predissolved in 20 ml of water is added, followed by addition of 0.0018 mol of potassium persulfate (KPS) dissolved in a minimum amount of water. The mixture is stirred for 3 hours. The solution is then concentrated and then freeze-dried.

[0120] To isolate the polymer, a precipitation is performed according to the following procedure:

[0121] The solid obtained is redissolved in 100 ml of methanol. The hydrochloride present is neutralized by adding 0.0054 mol of KOH (i.e. 0.30 g dissolved in about 25 ml of methanol) incorporated dropwise into the solution. The salt formed, KCl, precipitates and is extracted by filtration. The filtrate thus recovered is concentrated and then poured dropwise into 4 liters of ether. The precipitated polymer is recovered by filtration through a No. 4 sinter funnel. The solid is then dried under vane-pump vacuum. The mass yield is about 50%.

[0122] The above protocol leads to an amino polymer known as “PDMA” and corresponds to initiator/monomer ratios Ro=0.03 and Ao=0.01, in which:

[0123] Ro=[R—SH]/[NIPAM] and Ao=[KPS]/[NIPAM].

[0124] 2) Modification of the Amino PDMA

[0125] The PNIPAM macromolecules synthesized contain amine functions at the chain ends, these chains originating from the initiator aminoethanethiol AET.HCl.

[0126] By reaction of the amine function with acrylic acid, a vinyl double bond is attached to the chain end according to the following reaction scheme:

[0127] Procedure:

[0128] 50 ml of methylene chloride, 1.5 g of acrylic acid (0.021 mol), 9 g of PDMA and 4.3 g of dicyclohexylcarbodiimide (DCCI) (0.021 mol) are placed in a 100 ml beaker.

[0129] The reaction medium is stirred for one hour. Since the acrylic acid is in large excess relative to the PDMA (the amount of acrylic acid is about twenty times that of the PDMA), all the amino functions have been modified. The mixture is then filtered through a No. 4 sinter funnel to remove the precipitated dicyclohexylurea, the by-product resulting from the conversion of the DCCI. The purification is performed by precipitation from ether.

[0130] A macromonomer PDMA-1 bearing an allyl function at the chain end is thus obtained with a mass yield of about 70%.

[0131] The average molar mass and the polydispersity of the macromonomers thus prepared, measured by SEC (steric exclusion chromatography), are of the order of 15 000 and 2, respectively.

EXAMPLE 2

[0132] Preparation of a copolymer P(AM-PDMA)-1 with an acrylamide skeleton and PDMA grafts, of molecular mass 1 500 kdalton.

[0133] The copolymerization of amino PDMA (0.4 g) and of acrylamide (2.8 g) is performed for 4 hours in 50 ml of water at room temperature, while degassing vigorously with argon. The initiator used is the redox couple of ammonium persulfate ((NH₄)₂S₂O₈) [0.075 mol % of the amount of monomers]/sodium metabisulfite (Na₂S₂O₅) (0.0225 mol % of the amount of monomers). The resulting copolymer is purified by precipitation from acetone and dried under vacuum. Its molecular mass is 1 500 kdalton, and its polydispersity Mw/Mn is about 2. The degree of incorporation of macromonomer, measured by proton NMR, is about 6%, which corresponds to an average number of side branches on the skeleton of about 6.

[0134] On account of the free-radical polymerization method used, the macromonomers constituting the side chains are incorporated into the polymer chain at random positions determined by chance by the collisions between molecules (random distribution). This polymerization method leads to a distribution of the molecular masses of the polymer segments of the skeleton between two side branches of approximately exponential shape, and thus to polydispersities of said polymer segments of the skeleton that are largely superior to 1.8.

EXAMPLE 3

[0135] Separation properties obtained for single-stranded DNA (50-500 bp sizer, Pharmacia Biotech) at 50° C. in an ABI 310 machine (Perkin-Elmer), in a 100 mM Na TAPS, 2 mM EDTA, 7 M urea buffer, in various separation media. It is observed visually (FIG. 4) and more quantitatively by means of the resolution measurements (FIG. 5), that the separating medium according to the invention P(AM-PDMA)-2 improves the resolution relative to the same polymer skeleton not bearing side branches (PAM, FIGS. 2 and 5), but also relative to a commercial product based on linear PDMA (POP6, FIGS. 3 and 5). The separation time is also reduced, which is an additional advantage of the media according to the invention. It thus appears, surprisingly but beneficially, that this polymer according to the invention which comprises a large fraction of acrylamide, and a smaller fraction of PDMA, has, on account of the particular arrangement of said fractions and of the presence of junction points that characterize the invention, properties that are superior to those of each of said components in homopolymer form.

EXAMPLE 4

[0136] Preparation of a copolymer P(AM-PDMA)-2 containing an acrylamide skeleton and PDMA grafts, of molecular mass about 3 000 kdalton

[0137] The preparation is identical to that described in example 2, except for the concentration of ((NH₄)₂S₂O₈) [0.1 mol % instead of 0.075 mol % of the amount of monomers] and of (Na₂S₂O₅) (0.015 mol % instead of 0.0225 mol % of the amount of monomers). The viscosity, presented in FIG. 6, makes it possible to evaluate the molecular mass, of about 3 000 kdalton, starting from that of the p(AM-PDMA)-1, using the cubic dependence of the viscosity as a function of the molecular mass for interlocked polymers.

EXAMPLE 5

[0138] Preparation of a copolymer P(AM-PDMA)-3 bearing PDMA grafts, of molecular mass about 30 000.

[0139] In a first stage, the macromonomer of molecular mass 30 000 is prepared as described in example 1, with the exception of the ratio Ro, which is set at 0.015 instead of 0.03. This macromonomer is then polymerized with acrylamide, according to the protocol described in example 4.

EXAMPLE 6

[0140] Measurement of the viscosity of 3% solutions obtained with the polymers described in examples 2, 4 and 5, and also with a linear acrylamide homopolymer. In this example, each of the polymers was introduced at a rate of 3 g/100 ml into purified water (MilliQ). The viscosity of each of the corresponding solutions was measured on a Brookfield DV3 cone-plate rheometer run by the Rheocalc software (Sodexim, Muizon, F). The shear rate selected is 10 (1/s) for a temperature gradient of 1° C. per minute. It is observed in FIG. 6 that the copolymers according to the invention have no thermosensitive nature (their viscosity shows a small and uniform decrease with temperature), and a moderate viscosity. It is also observed that the structure and properties of the copolymers can be varied by controlling the polymerization conditions.

EXAMPLE 7

[0141] Electrophoretic separations of single-stranded DNA fragments, in separating media according to the invention based on copolymers described in examples 2, 4 and 5, and, for comparative purposes, in linear polyacrylamide (LPA) and the commercial separating medium “POP5” (Applied Biosystems). The separation conditions are identical to those of example 4, with the exception of the sample, a “sizer” of 100 to 1 500 bases (BioVentures, USA). It is observed in FIG. 7 that in the range that is most advantageous for sequencing (fragment size 600 to 1 000), the media based on copolymers according to the invention, in particular those corresponding to a mass concentration in the separating medium of 3%, lead to a resolution that is markedly superior to that obtained with the polymers of the prior art. Considering that a resolution of the order of 0.3 to 0.5 is sufficient to sequence DNA to within one base, the media according to the invention should allow reading lengths of greater than 800 bases. 

1. A non-thermosensitive liquid medium for analyzing, purifying or separating species inside a channel, and comprising at least one polymer composed of several polymer segments, characterized in that said polymer is of the irregular block copolymer type or irregular comb polymer type and has on average at least three junction points established between polymer segments of different chemical or topological nature.
 2. The medium as claimed in claim 1, characterized in that all the segments of at least one type of chemical or topological nature forming part of the composition of said polymer have a polydispersity of at least 1.5.
 3. The medium as claimed in claim 1 or 2, characterized in that the segments of each of the types of chemical or topological nature forming part of the composition of said polymer have a polydispersity of at least 1.5.
 4. The medium as claimed in claim 2 or 3, characterized in that said polydispersity is greater than 1.8.
 5. The medium as claimed in any one of the preceding claims, characterized in that said polymer has an average molecular mass of greater than 50 000 and preferably greater than 300
 000. 6. The medium as claimed in any one of claims 1 to 5, characterized in that said polymer shows specific affinity for the walls of said channel.
 7. The medium as claimed in claim 6, characterized in that said polymer has at least one type of polymer segments showing, within the separating medium, specific affinity for the wall, and at least one type of polymer segment showing in said medium less or no affinity for the wall.
 8. The medium as claimed in claim 6 or 7, characterized in that all the segments showing specific affinity for the wall represent between 2% and 80% by mass of the average total molar mass of said polymer.
 9. The medium as claimed in one of the preceding claims, characterized in that the polymer shows specific affinity for one or more analytes.
 10. The medium as claimed in claim 9, characterized in that the polymer bears nucleotides or polypeptides of determined sequence.
 11. The medium as claimed in claim 9, characterized in that the polymer is combined with a protein, a protein fraction, a protein complex and/or an acidic or basic function.
 12. The medium as claimed in one of the preceding claims, characterized in that all the polymer segments of at least one type of chemical or topological nature have on average a number of atoms of greater than 75, and preferably greater than 210, or have a molecular mass of greater than 1 500 and preferably greater than 4
 500. 13. The medium as claimed in any one of the preceding claims, characterized in that the various types of polymer segments of which said polymer is composed have on average an average number of atoms of greater than 75, and preferably greater than 210, or have a molecular mass of greater than 1 500 and preferably greater than 4
 500. 14. The medium as claimed in any one of the preceding claims, characterized in that the polymer has on average a number of junction points of between 4 and
 100. 15. The medium as claimed in any one of the preceding claims, characterized in that the polymer is a block copolymer containing on average at least four polymer segments.
 16. The medium as claimed in any one of the preceding claims, characterized in that the polymer is a comb polymer containing on average at least two side chains.
 17. The medium as claimed in any one of the preceding claims, characterized in that the polymer comprises at least one type of segment chosen from polyethers, polyesters, for instance polyglycolic acid, soluble random homopolymers and copolymers of the polyoxyalkylene type, for instance polyoxypropylene, polyoxybutylene or polyoxyethylene, polysaccharides, polyvinyl alcohol, polyvinylpyrrolidone, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyoxazoline, polystyrene sulfonate, and substituted or unsubstituted acrylamide, methacrylamide and allyl polymers and copolymers.
 18. The medium as claimed in any one of the preceding claims, characterized in that said polymer comprises at least one polymer chosen from: copolymers of the comb copolymer type, the skeleton of which is of dextran, acrylamide, acrylic acid, acryloylaminoethanol or (N,N)-dimethylacrylamide type and onto which are grafted side segments of acrylamide, substituted acrylamide or (N,N)-dimethylacrylamide (DMA) type, or of the DMA/allyl glycidyl ether (AGE) copolymer type, or of homopolymer or copolymer of oxazoline or of oxazoline derivatives; non-thermosensitive copolymers of the irregular sequential copolymer type having along their skeleton an alternation of segments of polyoxyethylene type and of segments of polyoxypropylene type, or an alternation of segments of polyoxyethylene type and of segments of polyoxybutylene type, or an alternation of segments of polyethylene and of segments of polyether type that are more hydrophobic than polyoxyethylene; copolymers of the irregular sequential block copolymer type having along their skeleton an alternation of segments of acrylamide, acrylic acid, acryloylaminoethanol or dimethylacrylamide type, on the one hand, and segments of (N,N)-dimethylacrylamide (DMA) type, or of DMA/allyl glycidyl ether (AGE) copolymer type, or of homopolymer or copolymer of oxazoline or of oxazoline derivatives; polymers of the irregular comb polymer type, the skeleton of which is of the agarose, acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type, and onto which are grafted side segments of agarose, acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type; copolymers of the irregular comb copolymer type, the skeleton of which is of the acrylamide, substituted acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer type, of oxazoline and oxazoline derivative, of dextran, of agarose, of methylcellulose, of hydroxyethylcellulose, of modified cellulose, of polysaccharide or of ether oxide type, and bears short-chain hydrophobic side segments such as alkyl chains, aromatic derivatives, fluoroalkyls, silanes or fluorosilanes.
 19. The use of a medium as claimed in one of claims 1 to 18, for separating, purifying, filtering or analyzing species chosen from molecular or macromolecular species, biological macromolecules of nucleic acid type, their synthesis analogs, proteins, polypeptides, glycopeptides and polysaccharides, organic molecules, synthetic macromolecules or particles such as mineral particles, lattices, cells or organelles.
 20. The use as claimed in claim 19 or the use of a medium as claimed in one of claims 1 to 18, in a channel of which at least one dimension is submillimetric.
 21. The use as claimed in claim 19 or 20 or the use of a medium as claimed in one of claims 1 to 18, for electrokinetic separations.
 22. The use as claimed in one of claims 19 to 21 or the use of a medium as claimed in one of claims 1 to 18, for diagnosis, gene typing, and high-throughput screening, quality control, or for detecting the presence of genetically modified organisms in a product.
 23. A process for separating, analyzing and/or identifying species contained in a sample, characterized in that it comprises a/ the filling of the channel of a separating device with a separating medium as claimed in one of claims 1 to 18, b/ the introduction of said sample containing said species into one end of said channel, c/ the application of an external field intended to move certain species contained in the sample, and d/ the recovery of said species or the detection of their passage at a point along the channel that is different from the point of introduction of the sample. 